(1) Field of the Invention
The present invention relates to a Canis sphingosine-1-phosphate (S1P) receptor isoform 1 (cS1P1), the nucleic acid encoding the cS1P1 receptor, and methods for using the cS1P1 receptor and the nucleic acid encoding the cS1P1 receptor in assays for identifying analytes which modulate activity of the cS1P1 receptor. The assays are useful for identifying analytes with immunosuppressive activities.
(2) Description of Related Art
Sphingosine-1-phosphate is a bioactive lysolipid that mediates a variety of diverse cellular functions such as cell adhesion, motility, differentiation, proliferation, and survival (Pyne and Pyne, Biochem. J. 349: 385-402 (2000); Hla, Pharmacol. Res. 47: 401-7 (2003); Spiegel and Milstien, Nat. Rev. Mol. Cell. Biol. 4(5): 397-407 (2003)). It is a metabolic product of sphingolipids which are ubiquitous phospholipids found in all eukaryotic cells. It is also an abundant blood lipid secreted by hematopoietic cells and released from activated platelets (Yatomi et al., J. Biochem. 121: 969-73 (1997)). Many of its cell signaling functions occur through activation of a family of G protein coupled receptors (GPCRs). Five sphingosine 1-phosphate (S1P) activated GPCRs have been identified, S1P1, S1P2, S1P3, S1P4, and S1P5 (previously known as endothelial differentiation genes Edg1, Edg5, Edg3, Edg6, and Edg8, respectively). These S1P receptors have a widespread cellular and tissue distribution and are well conserved in human and rodent species (Spiegel and Milstien, Biochim. Biophys. Acta 1484: 107-16 (2000); Fukushima et al., Annu. Rev. Pharmacol. Toxicol. 41: 507-34 (2001); Hla, Prostaglandins Other Lipid Mediat. 64(1-4): 135-42 (2001)).
Each S1P receptor has a unique tissue expression pattern and couples to a distinct set of heterotrimeric G proteins (Gα, Gβ, and Gγ) each of which leads to activation of an isoform-specific panel of multiple intracellular signaling pathways. Each S1P receptor is a transmembrane protein comprising a ligand binding domain, seven transmembrane domains, and a cytoplasmic domain which interacts with Gα of the set of heterotrimeric G proteins. In the inactive state, Gα is bound to GDP. When sphingosine-1-phosphate binds to the ligand binding domain, a signal is transduced through the S1P receptor which results in the GDP bound to Gα to be replaced by GTP and the Gα to dissociate from Gβ and Gγ (which remain as a GβGγ dimer). Gα and the GβGγ dimer activate effectors which in turn activate distinct intracellular pathways specific to the receptor and G protein. At present, five different Gα proteins subtypes are known; they are Gs, Gi/o, Gq, G12, and G13. Gs activates adenyl cyclase, Gi/o inhibits adenyl cyclase, and Gq activates phospholipase C beta (PLC) which cleaves phosphoinositol-4,5 bisphosphate (PIP2) in the cell membrane to release second messengers diacylglycerol (DAG) and inositol-(1,4,5)-triphosphate (IP3). G12 and G13 interact with Rho-specific guanine nucleotide exchange factors and regulate the actin cytoskeleton. The SIP1 receptor is coupled primarily via Gi/o to inhibit adenylate cyclase and stimulate mitogen-activated protein kinase (MAPK). It is also coupled to stimulate PLC via a PTX-sensitive Gi/o. The SIP2 receptor is coupled via Gi/o to Ras/MAPK like S1P1 but unlike S1P1, S1P2 is also coupled to stimulation of PLC via a PTX-insensitive Gq. It is also coupled to Rho stimulation. S1P3 also interacts with multiple Gα subtypes including Gi/o, Gq, and G12/13, whereas S1P4 primarily activates Gi/o and the MAPK pathway. The SIP5 receptor is coupled via Gi/o and G12 to inhibit adenylate cyclase in a PTX-sensitive manner but unlike S1P1, it does not stimulate MAPK.
Many of the physiological functions of sphingosine 1-phosphate and its receptors have now been elucidated. The S1P1 receptor is required for vascular maturation in mice (Liu et al., J. Clin. Invest. 106: 951-61 (2000)). Ligand-induced activation of S1P1 and S1P3 on endothelial cells has been shown to promote angiogenesis, chemotaxis, and adherens junction assembly through Rac and Rho (Lee et al., Cell 99: 301-12 (1999)). Lung endothelial barrier function is enhanced by sphingosine 1-phosphate activation of the S1P1 receptor (Schaphorst et al., Am. J. Physiol. Lung Cell Mol. Physiol. 285: L258-67 (2003)) whereas endothelial cell permeability is increased by activation of the S1P2 receptor (Schaphorst et al., Am. J. Physiol. Lung Cell Mol. Physiol. 285: L258-67 (2003)). S1P activation of the S1P2 receptor also inhibits chemotaxis by blocking Rac activation (Sugimoto et al., Mol. Cell. Biol. 23(5): 1534-45 (2003)) and promotes neurite retraction (Van Brocklyn, et al., J. Biol. Chem. 274(8): 4626-32 (1999)). Cardiovascular effects have been measured for sphingosine I-phosphate in rats and in dog hearts (Sugiyama et al., Jpn. J. Pharmacol. 82: 338-42 (2000); Sugiyama et al., Cardiovasc. Res. 46: 119-25 (2000); Yatomi et al., J. Biochem. 121: 969-73 (1997); Forrest et al., J. Pharm. Exp. Therap. 309: 758-768 (2004)); the S1P2 receptor has been implicated in contraction of coronary arteries (Ohmori et al., Cardiovasc. Res. 58: 170-7 (2003)); and, the S1P3 receptor has been found to mediate vasoconstriction of cerebral arteries (Salomone et al., Eur. J. Pharmacol. 469: 125-34 (2003)) and induce bradycardia and hypertension in rodents (Forrest et al., J. Pharm. Exp. Therap. 309: 758-768 (2004)). Pharmacological agonists of the S1P receptors are immunosuppressive; they regulate leukocyte trafficking by sequestering lymphocytes in secondary lymphoid organs (Brinkmann et al., J Biol. Chem. 277: 21453-7 (2002); Mandala et al., Science 296: 346-9 (2002). While the functions of the S1P4 and S1P5 receptors are less well understood, the S1P4 receptor has been shown to be localized to hematopoietic cells and tissues (Graeler et al., Curr. Top. Microbiol. Immunol. 246: 131-6 (1999)) and the S1P5 receptor has been shown to be primarily a neuronal receptor with some expression in lymphoid tissue in rodents but with a broader, expression pattern in human tissues (Im et al., J. Biol. Chem. 275(19): 14281-6 (2000); Niedernberg et al., Biochem. Pharmacol. 64: 1243-50 (2002)).
In light of the above, there is a need for methods for identifying analytes which modulate the activity of the S1P1 receptor.